In vitro DNA synthesis systems based on plasmid DNA species would be used to study the initiation and termination processes in bacterial DNA replication. Origin-plasmids have been constructed using P1 transduction in E.coli and using transformation of E.coli with restriction fragments of S.typhimurium and E.coli F-prime plasmids, with and without the plasmid vehicle pTK16. These plasmids would be further analyzed 1) to generate smaller origin-plasmids, with possible DNA sequencing of the oriC of S.typhimurium and E.coli, and 2) to obtain a detailed restriction and genetic map of the origin regions of these chromosomes. Small RNA species have been isolated and would be characterized using the origin-plasmids. Results from our previous in vivo initiation studies would be used to establish in vitro DNA initiation systems, using origin-plasmid DNA and concentrated cell lysates. These systems would be used 1) to isolate and characterize the dnaA gene product, 2) to study regulation of initiation, and 3) to characterize origin-RNA species synthesized in vivo and in vitro. Radioactivity experiments using E.coli dnaB252 would yield a restriction map of the termination region (ter locus). Restriction analysis of E.coli F-prime F123 which carries the ter locus would permit isolation and cloning of the ter locus, and deduction of a precise restriction and genetic map of the termination region. Chimera plasmids carrying the bacterial oriC origin and the E.coli ter locus would be constructed, to study termination in vitro. The primary cell target for transformation of a cell into a malignant cell is the cellular DNA. Further, the overall rate of chromosome duplication is controlled primarily by the initiation process, and chromosome segregation by the termination process. This project would thus study the molecular basis of that part of the DNA replication process, namely, initiation and termination, most relevant to the cancer problem, demonstrating its direct relevance to the health sciences.